Arabinogalactan proteins
Pbio691 - Plant Cell Wall
11/05/2010
Laura Cristea
AGPs
Plant primary cell wall
 Cellulose
 Hemicellulose
 Proteins
AGPs
Overview
 HRGPs
 Lowest protein content among HRGPs (1-10%)
 Highest sugar content among HRGPs (90-99%)
 Complex glycosylation modules
 Protein backbone O-glycosylated Ara, Gal, Rha, GlcUA, Fuc
 Soluble or GPI-anchored
 Associated with plasma membrane, cell wall
 Regulation, signaling, growth and development
 Cell-cell interaction, pathogen defense
 Substrate for pollen growth, wound-induced (gum arabic)
AGPs
Classification based on
structure
 Classical Hyp-rich AGPs (A)
 Classical AGPs with Lys-rich domain (B)
 AG peptide (12 aa) (C)
 Nonclassical AGPs with Asn-rich domain (D)
 Proteins with two AGP and two fasciclin domain (E)
 Proteins with two AGP and one fasciclin domain (F)
 Proteins with one AGP and one fasciclin domain (G)
Albersheim, P. et al. Plant Cell Walls (2010)
AGPs
Gum arabic glycoprotein
 Gum
arabic
 Ala-poor, His-rich
 Extensin motif
 Intermediate between AGPs and extensins
 Repetitive consensus motif
Ser-Hyp-Hyp-Hyp-Thr-Leu-Ser-Hyp-Ser-Hyp-Thr-Hyp-Thr-Hyp-Hyp-Leu-Gly-Pro-His
 Sugar composition resembles the AGPs with arabinose and galactose as major ones
AGPs
Biosynthesis
 Secretory
pathway
 Hydrophobic C-terminal – GPI
 Ala, Thr, Ser, Pro, Hyp rich
 ER – protein backbone
 Prolyl hydroxylase – not all Pro
 Golgi – Hyp-O-glycosylation –not all Hyp
 Glycosyltransferases
 Hyp glycosylation hypothesis
 beta glucosyl Yariv reagent – identification
problems
Buchanan, Gruissem, Jones Biochemistry & Molecular Biology of Plants
AGPs
Prolyl hydroxylase
 Post-translational
 Type II integral membrane protein
 Affinity – > 4 Pro residues
 Atmosferic oxygen needed
 O of 4-OH from Hyp – oxygen
Albersheim, P. et al. Plant Cell Walls (2010)
AGPs
Hyp contiguity hypothesis
 Contiguous
Hyp residues are arabinosylated (extensins)
 Noncontiguous Hyp are not glycosylated or just have one arabinose
 Clustered Hyp residues are linked to a galactose backbone with arabinose and
galactose as major components in the side chains; other types of sugar might be
present
Conclusion: the glycosylation pattern of the AGP protein backbone is
determined by the amino acid sequence.
AGPs
Enzymes for degradation
http://www.molbiol.saitama-u.ac.jp/bussitsu/research.html
AGPs
O-glycosylation in general
Wilson, Iain BH (2002) Curr. Opinion in Structural Biology 12, 569-577
Plant O-Hydroxyproline Arabinogalactans
Are Composed of
Repeating Trigalactosyl Subunits with
Short Bifurcated Side Chains
Li Tan, Peter Varnai, Derek T.A. Lamport,
Chunhua Yuan, Jianfeng Xu, Feng Qiu,
Marcia J. Kieliszewski
J. of Biol. Chem. Vol. 285, no. 32, 24575-24583 (2010)
Gene design
(AP)51
IFNα2-(SP)20
Subcloning for gene expression
Tobacco extensin signal sequence
CaMV 35S
Plant transformation vector pBI121
Agrobacterium LBA4404
transformation
Tobacco
Tobacco
suspension cells
suspension cell
transformation
culture
Protein separation
Protein biochemical
analysis
NMR structure
determination
Gene design
(AP)51
IFNα2-(SP)20
Gene design
Tan, L. et al. (2003) Plant Physiology 132, 1362-1369
IFNα2-(Ser-Hyp)20
Note: IFNα2 sequence not detailed
Xu, J. et al. (2007) Biotechnology & Bioengineering
Protein separation
Protein biochemical
analysis
Tobacco cells
media
Hydrophobic
Interaction
Chromatography
(HIC)
Hyp-arabinogalactan
(Ala-Hyp)51-EGFP
Cation & Size exclusion
chromatography
INFα2-(Ser-Hyp)20
Isolation & purification
 NaOH hydrolysis (108°C, 18 h)
 Separation on size-exclusion chromatography (Superdex-Peptide column)
 Hyp analysis - colorimetric
 Monosaccharide analysis
 Total sugar content – colorimetric (anthrone method)
 Neutral sugar content – gas chromatography (alditol acetates method)
 Nuclear Magnetic Resonance (NMR)
 INF Hyp-polysaccharide 1
 AHP-1
AHP-1
tube 23
Tan, L. et al. (2004) The Journal of Biological Chemistry 279:13, 13156-13165
NMR spectroscopy
One-dimensional 1H
Two-dimensional 1H homonuclear
 COrrelation SpectroscopY (COSY)
 TOtal Correlation SpectroscopY (TOCSY)
 ROtating Frame NOE SpectroscopY (ROESY)
 Nuclear Overhausser Effect SpectroscopY (NOESY)
Two-dimensional 13C, 1H
 Heteronuclear Single Quantum Coherence (HSQC)
 Heteronuclear Multiple Bond Coherence (HMBC)
Two dimensional 13C, 1H heteronuclear HSQC-TOCSY
 Two dimensional 13C, 1H HSQC—NOESY
NMRPipe
NMRView
 Standard: 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS)
Chemical shifts of 1H & 13C
Gane, A.M. et al. (1995) Carbohydrate Research 277, 67-85
1H
NMR, HSQC, HSMB
from a previous paper
One dimensional 1H – AHP-1
A:B:C:D:E:F:G = 4:1:1:1:4:1:4
A – Ara
D – Hyp H-4
B – Ara
E – Gal
C – Rha
F – Gal
G – Gal & GlcA
Tan, L. et al. (2004) The Journal of Biological Chemistry 279:13, 13156-13165
HSQC & HMBC
INF Hyp-polysaccharide 1
Sugar ratio & configuration
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
Sugar ratio & configuration
1H
NMR
A:B:C:D:E:F:G = 6:2:2:1:5:1:4+2
A – Ara
D – Hyp H-4
B – Ara
E – Gal
C – Rha
F – Gal linked to Hyp
G – Gal + GlcUA
INF Hyp-polysaccharide 1
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
TOCSY
Hyp-Gal linkage
HSQC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
Hyp-Gal linkage
HMBC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
1H
NMR
Gal configurations
HMBC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
TOCSY
Rha residues
HSQC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
Rha – GlcUA
GlcUA - Gal
HMBC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
1H
NMR
Ara linkages
HMBC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
HSQC
Ara linkages
HMBC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
INF Hyp-polysaccharide 2
Sugar composition
1H
NMR
Gal:Ara:GlcUA:Rha – 10:5:4:1
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
Gal linkage & backbone
HMBC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
1H
NMR
Side chains
HMBC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
Primary structures
IFN-Hyp polysaccharide 1
IFN-Hyp polysaccharide 2
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583
Conclusions
 Complete structure elucidation by NMR
 INF Hyp-polysaccharide 1 has six-residue galactan chain with 2 beta 1,3
linked by a beta 1,6 linkage
 INF Hyp-polysaccharide has four side chains
 Repetitive trisaccharide with two six-residue bifurcated side chains
 Six-residue side chain – identical with gum arabic side chain (no ter 5Ara)
 Glycosylation is not determined by the non-glycosylated sequence or
type of peptide
 Incomplete glycosylation
Molecular Modeling